Cancer-associated fibroblasts actively compress cancer cells and modulate mechanotransduction

During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce an excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule acts as a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP and a decrease in proliferation. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.


Supplementary Data Figure. 3. Characterization of primary CAFs and cancer cells a,
Representative (from N=5 mice) image of a subcutaneous tumor stained for αSMA (cyan) and DNA (DAPI).Cancer cells express nuclear GFP (green) and the stroma express mTomato (magenta).Scale bar, 100 µm b, Flow cytometry analysis of the expression of CD31, CD45, Vimentin and EpCAM in primary cancer cells, and of CD31, CD45, EpCAM and αSMA in primary CAFs.The percentages (%) of stained cells (red) are plotted relative to the basal signal of an isotype control (light blue).c, Representative images of primary CAFs stained for F-Actin (phalloidin, magenta), αSMA (cyan) and DNA (DAPI, blue).N=2 independent experiments.Scale bar, 50 µm.In this note, we develop a physical model of the remodeling of the cancer cell cluster (CC cluster) by Cancer Associated Fibroblasts (CAFs) in the in vitro co-culture system.We aimed at providing a unified framework to quantitatively described the closure kinetics and the associated traction forces measured on the substrate, and to relate these to the pillar compression experiments.

S1 Model
A sketch of the model system is presented in Supp.Fig. 1a.The CC cluster constitutes a cell monolayer prior to CAF remodeling.The closure of the CAF monolayer on top of the cluster is assumed to be solely driven by the contraction of the acto-myosin ring present at the inner edge of the CAFs, modeled as a constant line tension Γ [1].We concentrate on the dynamics characterized by inward traction forces, for which CAF migration may be disregarded.We further assume radial symmetry, which, although not particularly accurate in experimental situations such as the one shown in Fig. 1d of the main text, is a prerequisite for an analytically tractable model.
We adopt a thin sheet approximation for the mechanical description of the plane stress tensor γ ij in the CAF monolayer, and the stress tensor in the CC cluster, for which we only consider the integrated version over cluster thickness hσ ij ≡ h 0 dz σ ij .Several spatial regions must be distinguished, characterised by the radius of the CAF inner edge (the acto-myosin ring) R c and the outer edge of the CC cluster R T (see sketch on Supp.Fig. 1a).
• For r > R T , the CAFs are in direct contact with the substrate and experience a substrate force density T r .The local force balance in this region reads [2]: where γ rr and γ θθ are the radial and orthoradial components of the CAF stress tensor and r is the radial coordinate.
• For R c +δ < r < R T , where δ is the width of the actomyosin ring, the CAFs are in contact with the CC cluster.The sliding of the CAFs on top of the CC cluster creates a shear stress f (r) which acts as a source of tension gradient in both CAFs and CC cluster: • For R c < r < R c + δ, the balance between the inward force from the ring line tension Γ/R c and the CAF radial tension may involve a particular lineic friction force f Γ , which in turn causes a discontinuity δhσ rr in the CC cluster stress : • For r < R c , the central part of the CC cluster is not covered by the CAFs.Prior to bud formation, the central cluster is weakly deformed and does not directly exert stress on the actomyosin ring.The force balance in the CC cluster simply reads The persistence of inward pointing traction forces after the ring has reached a stationary position (Fig. 1e, main text, reproduced in Supp.Fig. 1b) suggests an elastic description for the mechanics of CAFs and CC cluster, and an elastic-like interaction with the substrate [1,2].For a 3D-incompressible elastic description of the CAF monolayer characterised by a 2D elastic modulus E c , the components of the stress tensor are related to the radial displacement u(r) in the CAF layer (where r is the radial coordinate) according to: where γ c is the homeostatic contractile tension in the CAF monolayer far from the ring.Further assuming an elastic interaction with the substrate: T r (r) = Y s u(r) where Y s is a static friction coefficient, the solution of Eq. 1 for the CAF displacement in the region r > R T is u(r) ∝ K 1 (r/λ s ) where K 1 is a modified Bessel function, which leads to a spatial localisation of the substrate stress over a characteristic length scale λ s ≡ 4E c /(3Y s ) [2].This analytic solution is likely to be of limited validity in the present context, as the tissue mechanics is more complex than a simple elastic sheet, and include cellular rearrangements, especially during bud formation.However, the localisation of the traction forces outside the CC cluster is indeed observed (with λ s 50 µm), which suggests that the spatial derivative of the stress dominate the RHS of Eqs.1,2,4 (quasi 1D approximation).These three local force balances can be integrated and added to Eq. 3 to obtain the global force balance The LHS of this equation represents the force (per unit length) driving CAF closure and the RHS the forces resisting closure, which includes the CAF homeostatic tension, substrate friction, and the compression of the central region of the CC cluster.The expression given for γ bud corresponds to the low deformation limit of Eqs.1-4.However, Eq. 6 remains valid when a tumor bud forms in the central region tof the CC cluster, provided the bud compressive stress −γ bud is calculated by taking into account cellular rearrangement in the bud (see below).

S2 Traction force and closure dynamics
Eq. 6 may be seen as a dynamic equation for the evolution of the ring radius R c (t) as all the terms present in it (apart from γ c ) depend on time.The LHS of Eq. 6 clearly increases with time as the radius shrinks, so must the RHS.We may assume that the CAF tension far from the cluster γ c remains constant over time, so the increase of the closure force must be compensated by either an increase of traction force or by an increase of the bud resistance to compression or by both.Supp.Fig. 1c,d shows the temporal evolution of the total traction force1 and the average ring radius (obtained from the ring area shown Fig. 1f in the main text).Two dynamical regimes can be distinguished from the moment where the total traction force points inward (Supp.Fig. 1d).At earlier time (from 7 to 16 hrs), the closure dynamics is fast (dR c /dt = −3.3µm/h) and corresponds to a linear increase of the inward traction force with time.At later time (16 to 27 hrs), the closure dynamics is slower (dR c /dt = −1 µm/h) and the traction force saturates to a value R c ∞ 0 T r dr = 1.6µN.The linear increase of inward traction force at short time is consistent with the increase of the driving force from the line tension Γ/R c as the ring closes, although part of the increase of inward traction force must probably also be attributed to the decrease of active outward traction force associated to CAF migration.The fact that the traction force saturates while the ring is still closing is remarkable, and indicates a build-up of stress in the central cluster region that is eventually driving bud formation.In this regime, only a fraction of the driving force is compensated by the traction force, which, at their saturation value, amounts to an effective line tension Γ s 1.2 − 1.6µN.The remaining driving force must be compensated by the compression of the CC cluster.Thus, the CC cluster budding has a signature both in the closure dynamics and in the evolution of the traction force.
We can attempt to relate this value of the line tension to the stress measured in the pillar compression experiment reported in Fig. 4 -main text.We see from Fig. 4f that the normal stress on pillars of radius R p = 50 µm reaches σ p = 10 3 Pa.This corresponds to a typical lateral tension in the CAF layer surrounding the pillar γ p = σ p R p = 5 × 10 −2 N/m.Multiplying γ p by the typical thickness h c 10 µm of the CAF monolayer yields an estimate for the line tension Γ 500 nN.This is slightly smaller but of the same order of magnitude as the estimate obtained from the traction force in the co-culture experiments.This shows that the CAFs are indeed able to exert a large compressive stress, when compared, for instance, to the typical tension of one stress fiber, which is of order 10 nN [3].

S3 Bud stability
The cancer cell buds remain stable over time (Fig. 1e,f -main text and Supp.Fig. 1b) when directly compressed by the CAF ring.This experimental observation is not straightforward from a theoretical perspective.Indeed, the cellular rearrangements and multilayering involved in bud formation suggests a stress-dependent fluidification of the CC cluster.If the cluster behaved as a fluid, the bud would adopt a spherical shape due to its apical tension γ cc .Experimental observations show that buds are rather cylindrical with small height-to-diameter ratio (Extended Data Fig. 4b,c).Furthermore, the stability of such fluid bud pinched by the actomyosin ring would rely on the ratio of driving to resisting force Γ/(γ cc R c ) to be of order unity.With a typical CC apical tension γ cc ∼ 10 −3 N/m and with R c 100 µm, this ratio is about 10 times smaller.
To simplify the discussion of bud stability, we neglect the traction forces which saturate to a constant value after bud formation.We describe the bud as a stratified structure composed of n layers (Supp.Fig. 1e), and we only discuss the diagonal component of the stress tensor in each layer, defined as a pressure P .To account for the plastic cellular rearrangements within the bud, we introduce a yield pressure P * that describes phenomenologically the amount of compressive stress necessary to trigger cell exchange between layers.CAF closure initially occurs on a monolayer of CCs, and force transmission through friction (f (r) and f Γ in Eqs.2,3) leads to the generation of a compressive stress P 1 in the central part of the CC monolayer.If the pressure remains below the threshold (P 1 < P * ), no multilayering occurs and the CAFs are able to close on top of the CC monolayer, as it is sometimes observed in our experiments (data not shown).If the threshold is exceeded, cells delaminate from the central region of the CC cluster and form a second layer.The particular ring radius at which this happens is defined as R * c .The pressure P 2 (t) in the second layer increases as the ring closes and cells are transferred from the first layer.If P 2 > P * , a third layer forms, with pressure P 3 (t), and cells are expelled from the second payer until P 2 − P 3 < P * .Only the second layer is directly compressed by the ring, so the bud resistive force in Eq. 6 is γ bud = −hP 2 .The pressure P 2 is estimated as follows.For P 2 < P * , P , where E cc is the bulk modulus of the layer and R * 2 is the ring radius at which layer 2 starts being compressed (Supp.Fig. 1f ).For P 2 > P * , P 2 = P * φ(h b /h), where h b is the height of the bud, determined by volume conservation (h b = h(R * 2 /R c ) 2 ) and φ(x) is a phenomenological shape function capturing the shear stress between adjacent layers.In analogy with the standard problem of elastic pillar deformation induced by a collar pressure [4], we adopt the function φ(x) = 1 + b tanh[(x − 1)/b] which appropriately describes the localisation of the stress near the ring for tall bud (h b h), as seen in the pillar compression experiment (Fig. 4g -main text).Here, b is a phenomenological parameter representing shear resistance between adjacent layers.For an homogeneous elastic material, finite element simulations yield b 0.3.In the present context, b is determined by cell-cell adhesion and can be considered as a free parameter.
Supp.Fig. 1f shows a graphical representation of the static force balance between the ring tension and the bud compression Γ/R c = γ c + hP 2 .The existence of a static equilibrium with a finite ring radius state crucially depends on the parameter b.If the ring tension is shared by all layers (b → ∞), there is always a single stable equilibrium as the height of the bud diverges when R c vanishes (solid gray line in Supp.Fig. 1f ).In the opposite limit (b → 0) different layers are uncoupled, and P 2 ≤ P * .Since the formation of a second layer requires Γ/R c > γ c + hP * , no static equilibrium exists at finite R c and multilayering necessarily leads to ring closure below the bud (dashed gray line in Supp.Fig. 1f ).Under more realistic situations, bud compression leads to a maximum resistive force hP * (1 + b) that defines three phenotypes according to the parameter values (Supp.Fig. 1e,f ): full budding and CAF closure, stable bud with a finite neck size and CAF closure with no bud.This is summarized on Supp.Fig. 1g with a phase diagram as a function of reduced yield pressure P * /E cc and reduced line tension Γ/(E cc h 2 ).If P * is small, the CAF shear stress triggers the multilayering transition for large ring radius but the maximum bud resistive force is too small to equilibrate the ring compression.If P * is large, the multilayering transition occurs for a small ring radius, for which ring compression increases faster than the bud resistive force with decreasing ring radius.In both cases, cells are expelled from the second layer until the CAF ring closes.For even larger P * , multilayering would only occur for a ring radius of order the cell size, in which case we consider that the ring closes without bud formation (gray region on Supp.Fig. 1g).Thus, bud stability occurs only on a parametric region were both ring line tension and yield pressure are sufficiently small, and can be enhanced with an increase of CC elastic modulus E cc or effective parameter b.From the estimation Γ ∼ 1 µN obtained in the previous part and with h ∼ 10 µm, stable buds require E cc 1 kPa and P * 0.1 − 0.4E cc .Thus, CAF compression around an incomplete CC bud provides an indirect way to measure the tissue elastic modulus, found here to be consistent with typical values measured for cancer cells [5].

4 .
Characterization of primary CAFs by RNA sequencing a, Bubble plot representing the main gene ontology terms (KEGG) enriched when considering the top 100 expressed genes in wild type CAFs.b, Expression of selected gene sets in wild type CAFs, previously defined as markers for different CAF subtypes in pancreatic (iCAFs, apCAFs and myCAFs, Eylada et al. 2019) (left), and colorectal cancer (normal fibroblasts, CAFs subtype A and CAFs subtype B, Li et al. 2017) (right).Supplementary Data Figure.5. CAFs encapsulate and deform N/p53 tumor cell clusters a, Representative images (from n=68 positions, N=1) of CAFs cocultured with N/p53 tumor cells, evolving over time.Top panels, phase contrast.Lower panels, cancer cells (membrane-GFP, green) Red dashed lines delineate the contour of CAFs ring.White dashed lines delineate the contour of the tumor cell cluster.Scale bar, 100 µm.b, Representative (from n=14 positions, N=1) orthogonal images of CAFs cocultured with N/p53 tumor cells, evolving over time.Cancer cells (membrane-GFP, green) and CAFs (CellTracker, magenta).Scale bar, 100 µm.c, 3D rendering of CAFs-N/p53 tumor cells co-cultures after 24h in culture (from n=6 positions, N=1).Cancer cells (membrane-GFP, green) and CAFs (CellTracker, magenta), DNA (DAPI, cyan).Lower panel, orthogonal Z section.Scale bar: 50µm.d, Representative images (from n=4 positions, N=1) of CAFs and cancer cells that exited a PDX tissue fragment and spontaneously organized over time.Yellow dashed lines represent the CAFs edge and ring.Scale bar, 100 µm.Supplementary Data Figure.6. CAFs compress cancer cells using actomyosin contractility a, Schematic representation of the interpretation of force patterns generated by CAFs during cancer cells deformation and budding.Contraction of CAFs rings generate inward pointing forces (black arrows) that are transmitted through the CAFs monolayer to the polyacrylamide (PAA) gel (grey), generating a pattern of inward pointing radial tractions (blue arrows).b, Representative kymograph of circumferentially averaged radial (left) and tangential (right) tractions as a function of the distance to the center of the cancer cell cluster.Solid black line represents the cancer cell cluster contour.Outwards pointing radial tractions are defined as positive, and inwards pointing radial tractions are negative.Tangential tractions pointing clockwise are defined as positive, and tangential tractions pointing counterclockwise are negative.c, Circumferentially averaged tangential tractions Tt (purple) and radial tractions Tr (orange) as a function of the distance to the cancer cell cluster center after 36h of co-culture.Black dashed line represents the cluster boundary.Data represented as mean ± SD, n=49 clusters from N=3 independent experiments.d, Traction maps overlaid on a DIC image of cancer cell-CAFs after ≈40h co-culture (Pre-Y27632) and 6h after addition of Y27632 (6h+Y27632) (quantified in panel e).Solid red line represents the contour of CAFs ring; dashed line represents the contour of CAFs ring before Y27632 addition.Scale bar, 100 µm.e, Circumferentially averaged radial traction peak magnitude near the boundary of the cancer cell cluster (orange) and CAFs ring area (purple) normalized to the initial ring size, as a function of time.Dashed black line represents the moment of Y27632 addition.Data represented as mean ± SD, n=30 clusters from N=3 independent experiments.Supplementary Data Figure.7. Compression assay and single traction forces on normal fibroblasts and CAFs seeded on different stiffnesses.a, Top: Traction force maps (white vectors) overlaid with fibroblasts (magenta) and cancer cell clusters (green) 48 hours after seeding the fibroblasts.From left to right, fibroblasts are: CAFs control on 11kPa gels, CAFs control on 2kPa gels, NIH/3T3 normal fibroblasts on 11 kPa gels, and mouse normal intestinal fibroblasts on 11kPa.Scale bar 100 µm.Bottom: Radial and tangential traction profiles as a function of the distance to the cluster center for the different conditions.Vertical dashed line indicates the boundary between the cancer cells and the fibroblasts.Outwards pointing radial tractions are defined as positive, and inwards pointing radial tractions are negative.Tangential tractions pointing clockwise are defined as positive, and tangential tractions pointing counterclockwise are negative.Right graph: radial traction peak in each condition, defined as the traction peak in the radial traction profile within a disk of ~50µm around the boundary between the cancer cells and the fibroblasts.Data represented as mean ± SD. n=25 clusters with CAFs 11 kPa, n=25 clusters with CAFs 2 kPa, n=25 clusters with NIH/3T3, and n=21 clusters with intestinal fibroblasts, from N=2 independent experiments.b, Representative traction maps overlaid on CellTracker-labelled control CAFs, NIH/3T3 and mouse normal intestinal fibroblasts.Solid red line represents the cell contour.Scale bar, 50 µm.Scale vector, 400 Pa.Right dot plots, quantification of cell area and strain energy.Data represented as mean ± SD. n= 30 cells from all experimental conditions from N=2 independent experiments.Supplementary Data Figure.8. MyosinIIA KO CAFs do not exert traction forces or deform cancer cells a, Representative images (from n=7 images, N=1 independent experiment) of control (upper panels) and MyosinIIA KO (lower panels) CAFs after 7 days of in vitro knockout induction with 4-hydroxytamoxifen.Control CAFs (membrane tdTomato, magenta), KO CAFs (membrane-GFP, green, and low levels of membrane-tdTomato, magenta).Cells were stained for DNA (DAPI, blue) and MyosinIIA (right panels).Scale bar, 50 µm.b, Western blot for MyosinIIA (230KDa) in control and MyosinIIA KO CAFs after 4, 7 and 10 days of knockout induction.GAPDH (37KDa) was used as loading control (N=1).c, Representative traction maps (quantified on right dot plots) overlaid on images of control (membrane-tdTomato) and MyosinIIA KO (membrane-GFP) single CAFs after 7 days of in vitro knockout induction.Solid red line represents the cell contour.Scale bar, 50µm.Scale vector, 400 Pa.Right dot plots, quantification of mean traction and area per cell.Data represented as mean ± SD. n=71 cells, from N=3 independent experiments.*** p<0.001,Mann Whitney two-tailed test.d, Representative traction maps overlaid on a DIC image of a cancer cell and control (left) or myosin IIA KO (right) CAFs after 48h of culture (quantified in panel e).Black line represents the contour of the cancer cell cluster Scale bar, 100 µm.e, Circumferentially averaged radial tractions Tr in co-cultures of cancer cells and control (magenta) and MyosinIIA KO (orange) CAFs after 48h, as a function of the distance to the cancer cell cluster center.Black dashed line represents the cluster boundary.Data represented as mean ± SD.Control, n=36 clusters, MyosinIIA KO, n=30 clusters, from N=2 independent experiments.f, Fluorescence intensity as a readout of proliferation in control and Myosin IIA KO CAFs from N=2 independent experiments.Supplementary Data Figure.9. Pillar compression depends on CAFs contractility a, Schematic representation of the methodological approach for pillar microfabrication.b, Mean normal and tangential traction magnitude in control pillars.n=18 pillars, from N= 3 independent experiments.*** p<0.001,Wilcoxon matched-pairs signed rank test.c, Representation of CAF normal tractions averaged across pillar height, on a representative pillar before (magenta) and after (blue) Y27632 treatment.Scale vector, 600 Pa.Right dot plot, quantification of mean normal traction for each pillar before and after Y27632.n=12 pillars, from N=2 independent experiments.Supplementary Data Figure.10.Fibronectin and N-cadherin knockdown does not affect force generation at single cell level a, Western blot for N-Cadherin in control and two independent Cdh2 SiRNA CAFs.GAPDH was used as loading control.N=1.b, Representative traction maps (quantified in right dot plot) overlaid on phase contrast images of control and SiRNA#1 Cdh2 single CAFs.Solid red line represents the cell contour.Scale bar, 50 µm.Scale vector, 400 Pa.Right dot plots, quantification of mean traction and area per cell.Data represented as mean ± SD.Control, n=28 cells, SiRNA#1 Cdh2, n=30 cells, SiRNA#2 Cdh2, n=30 cells from N=2 independent experiments.c, Representative images of control (upper panels) and MyosinIIA KO (lower panels) CAFs, after 7 days of in vitro knockout induction with 4-hydroxytamoxifen.Cells were stained for F-actin (Phalloidin, magenta), Fibronectin (Green) and DNA (DAPI, blue).Scale bar, 100µm.N=1.d, Western blot for Fibronectin in control and MyosinIIA KO CAFs after 4, 7 and 10 days of knockout induction.GAPDH was used as loading control.N=1.e, Left: Western blot for Fibronectin in control and SiRNA Fibronectin CAFs.GAPDH was used as loading control.N=1.Right: Representative traction maps overlaid on phase contrast images of control and fibronectin-KD CAFs (SiRNA#1).Solid red line represents the cell contour.Scale bar, 50 µm.Scale vector, 400 Pa.Right dot plots, quantification of mean traction and area per cell.Data represented as mean ± SD.Control, n=28 cells, SiRNA#1 Fn1, n=29 cells, SiRNA#2 Fn1, n=30 cells, from N=2 independent experiments.f, Traction maps (quantified in right plots) overlaid on a fluorescent image of CAFs (magenta) and cancer cells (green) 48 hours after seeding.CAFs were transfected with siRNA control (left) or siRNA#2 Fn1 (right).Graph: average radial traction profiles (left) and radial traction peaks (right) of CAFs siRNA control and siRNA#2 Fn1.Vertical dashed lines indicate the boundary between the cancer cells and the CAFs.Data are presented as mean ± SD. n=17 clusters for siRNA control and n=10 clusters for siRNA#2 fibronectin, from N=2 independent experiments.g, Fluorescence intensity as a readout of proliferation in CAFs siRNA control, siRNA#1 Fn1 and siRNA#2 Fn1, from N=2 independent experiments.Supplementary Data Figure.11.Cancer cells mechanosense substrate stiffness through YAP.Upper panel: staining of YAP, F-Actin and DNA in single cancer cells seeded in substrates of different rigidities (0,2 and 11 kPa), coated with 1µg/mL of collagen 1, and treated or not with blebbistatin.Scale bar: 10µm.Lower panel: quantification of YAP nucleus/cytoplasm ratio per cell.Data represented as mean ± SD. n= 68 cells for 0,2 kPa, n=71 cells for 11 kPa conditions from N=3 independent experiments.n= 32 cells for 0,2 kPa + Blebbistatin, n= 42 cells for 11 kPa + Blebbistatin, from N=2 independent experiments.Supplementary Data Figure.12. Changes in gene expression and CAF subtypes upon Myosin IIA KO and Fibronectin KD. a, Volcano plots showing the differentially expressed genes in Myosin IIA KO vs. control CAFs (thresholds, Fold Change = 2,-2; P value = 0.01).b, Bubble plot representing the main gene ontology terms (KEGG) enriched in downregulated (left) and upregulated (right) gene sets in CAFs Myosin IIA KO compared to control.c, Volcano plots of the differentially expressed genes in siRNA Fibronectin CAFs vs. SiRNA Control CAFs (thresholds, Fold Change = 2,-2; P value = 0.01).Fibronectin KD gene ontology is not shown because we considered the number of differentially expressed genes too low to perform gene ontology analyses.d, Expression of selected gene sets, previously defined as markers for different CAF subtypes in pancreatic (iCAFs, apCAFs and myCAFs, Eylada et al. 2019) (left), and colorectal cancer (normal fibroblasts, CAFs subtype A and CAFs subtype B, Li et al. 2017) (right), comparing control vs. Myosin KO CAFs, and SiRNA Control vs. Fibronectin-depleted CAFs.Correlations were tested by linear regression (p<0.001 for all comparisons).nuclear correlation.b, Tumor CAF (left) and cancer cell (right) density vs. YAP nuclear correlation.Correlations were tested by linear regression.Supplementary Data Figure.15.CAF compression decreases the number of cancer cells in S-phase.a, Cancer cells (Cell tracker, Green) cultured on an adherent substrate where they grow freely, under confinement (PDMS stencils), or surrounded by control or Myosin IIA KO CAFs (Cell tracker, magenta).White lines represent the contour of the cluster of cancer cells.A 60 minutes pulse of EdU was performed before fixation to label cells in S-phase.Cells were then stained for incorporated EdU (yellow) and DNA (DAPI, blue).Graph: percentage of EdU + cells in the cancer cell cluster.Data represented as mean ± SD, n=8 clusters per condition, from N=2 independent experiments.Scale bar: 100 µm.b, Fluorescence intensity as a readout of proliferation in cancer cells treated with control or conditioned medium from control and MyosinIIa KO CAFs from N=2 independent experiments.